1. Field of the Invention
The present invention relates to mutant proteinase inhibitors and fragments thereof, including serpins (serine proteinase inhibitors), particularly inhibitory serpins such as plasminogen activator inhibitors (PAIs), anti-thrombin III, and α1-antitrypsin (α1AT). The proteinase inhibitors in question comprise an amino acid sequence with at least one epitope in which a mutation has altered the binding of the mutant molecule to an anti-proteinase inhibitor antibody, relative to the binding of a corresponding wild-type molecule to the same antibody. The invention also relates to the use of a mutant proteinase inhibitor or fragment thereof for screening or designing proteinase inhibitor-inactivating agents or compounds that have potential as therapeutic agents to treat conditions associated with increased levels of proteinase inhibitors in vivo. The invention more broadly relates to the use of such technology to identify tertiary binding epitopes in metastable proteins that are not amenable to rational drug design screening, thereby to screen for and design inhibitor compounds or agents that have potential to reduce levels of such metastable proteins in the body.
2. Description of the Related Art
Considerable information is available on proteinase inhibitors and their ability to regulate many biologically important systems in the body. The influence of these inhibitors is based on their ability to regulate a variety of divergent proteinases. Particularly important proteinase inhibitors are serpins, a super family of inhibitors that apparently share a common tertiary structure, Doolittle (1983), Science 222: 417-419, and may have a common evolutionary ancestor. Hunt et al. (1980), Biochem.Biophys.Res.Comm. 95:864-871. Proteins with recognizable sequence homology have been identified in vertebrates, plants, insects and viruses but not, thus far, in prokaryotes. Huber et al., (1989). Biochem. 28: 8951-8966; Sasaki (1991), Eur.J.Biochem. 202:255-261; Komiyama et al. (1994) J.Biol. Chem. 269: 19331-19337. Current models of serpin structure are based largely on seminal X-ray crystallographic studies of one member of the family, α1-antitrypsin (α1AT). Huber et al., (1989),. supra. Loebermann and co-workers solved the structure of a modified form of α1AT, cleaved in its reactive center, by proposing a model where the native reactive center is part of an exposed loop, also called the strained loop. See Loebermann et al., (1984). J.Mol.Biol. 177: 531-557; Carrell et al. In PROTEINASE INHIBITORS 403-20 (Elsevier Science Publishers, 1986); Sprang, S. R. (1992). Trends Biochem.Sci. 17: 49-50.
Recent crystallographic structures of several native serpins, with intact reactive center loops, are consistent with Loebermannø hypothesis that the overall native serpin structure is very similar to cleaved α1AT, but that the reactive center loop is exposed above the plane of the molecule. Schreuder et al., (1994) Nature Structural Biology 1: 48-54; Carrell et al. (1994) Structure 2: 257-270; Stein et al. (1990) Nature 347: 99-102; Wei et al., (1994) Nature Structural Biology 1: 251-258.; Sharp et al., (1999) Structure 7:111-118. Additional evidence has come from studies where synthetic peptides, homologous to the reactive center loops of α1AT, antithrombin III (ATIII), or plasminogen activator inhibitor-1 (PAI-1), when added in trans, incorporate into their respective molecules, presumably as a central strand of β-sheet A. Björk, et al. (1992), J.Biol.Chem. 267, 19047-19050; Björk, I. (1992), J.Biol.Chem. 267, 1976-1982; Schulze et al. (1990), Eur. J. Biochem. 194: 51-56; Carrell et al. (1991), Nature 317:730-732; Kvassman et al. (1995) Bichem. 37: 15491-15502. This leads to an increase in thermal stability similar to that observed following cleavage of a serpin at its reactive center, and converts the serpin from an inhibitor to a substrate for its target proteinase.
An additional serpin structural form has also been identified, the so-called latent conformation. In this structure the reactive center loop is intact, but instead of being exposed, the entire amino-terminal side of the reactive center loop is inserted as the central strand into β-sheet A. Mottonen et al. (1992) Nature 355:270-273. This accounts for the increased stability of latent PAI-1 (Lawrence et al.(1994b) Biochem. 33: 3643-3648), as well as its lack of inhibitory activity (Hekman et al. (1985) J.Bio.Chem. 260:11581-11587). The ability to adopt this conformation is not unique to PAI-1, and has also now been shown for ATIII and α1AT. Carrell et al. (1994), supra; Lomas et al. (1995) J.Bio.Chem. 270:5282-5288. Together, these data have led to the hypothesis that active serpins have mobile reactive center loops, and that this mobility is essential for inhibitor function. Carrell et al. (1991), supra; Carrell et al. (1992), Curr.Opin.Struct.Biol. 2: 438-446; Lawrence et al. (1994a) J.Bio.Chem. 269:27657-27662; Shore et al. (1994) J.Bio.Chem. 270:5395-5398; Lawrence et al. (1995) J.Bio.Chem. 270:25309-25312; Fa et al.(1995) Biochem. 34: 13833-13840; Olson et al. (1995) J.Bio. Chem. 270:30007-30017; Lawrence et al. (1990) J.Bio.Chem. 265:20203-20301.
An important member of the serpin super-family is plasminogen activator inhibitor (PAI). The PAIs have become recognized as critical regulators of the plasminogen activator (PA) system. The identification of an efficient inhibitor of t-PA in endothelial cells was first reported by Loskutoff et al., (1983) Proc. Natl. Acad. Sci. USA 80:2956-2960. Four kinetically relevant PAIs are recognized currently: PAI-1, initially described as the oendothelial cell PAI,p PAI-2, also referred to as oplacental PAI,p PAI-3, also known as oactivated protein C-inhibitor,p and proteinase nexin 1 (PN-1), also called oglia-derived neurite-promoting factor.p
Recent interest has centered on PAI-1 because it plays an important role in fibrinolysis and is an established risk factor for cardiovascular disease. PAI-1 is the major plasminogen activator (PA) inhibitor in plasma and platelets. Booth et al., (1988) Br.J.Haematol. 70:327-333; Fay et al.(1992) N.Engl.J.Med. 327:1729-1733; Fay et al.(1994) Blood 83:351-356.
The PAI-1 gene is 12.3 kb in length, and yields two mRNA species of 2 kb and 3 kb that both encode the same 50 kDa single-chain glycoprotein. Ny et al. (1986)) Proc. Natl. Acad. Sci. USA 83:6776-6780; Strandberg et al. (1988), Eur. J. Biochem. 176: 609-616; van Mourik et al. (1984) J.Bio.Chem. 259:14914-14921. PAI-1 is the most efficient inhibitor known of both uPA and tPA. Lawrence et al. (1989), Eur. J. Biochem. 186: 523-533; Sherman et al. (1992) J.Bio.Chem. 267:7588-7595.
PAI-1 exists in three interconvertible conformations: an active, a latent and a substrate form. The active conformation inhibits its target proteinases, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), by the formation of stable covalent complexes. The reactive site bond (P-P′1) is inaccessible to the target proteinases in the latent form whereas the noninhibitory substrate form of PAI-1 is cleaved at the reactive site bond by the serine proteinases, resulting in an irreversible inactivation of PAI-1, and with the regeneration of the proteinase activity.
Active PAI-1 decays to the latent form with a half-life of approximately 1 hour at 37° C. With exposure to denaturants (guanidine HCl or SDS), latent PAI-1 can be returned partially to the active form. Though recent X-ray crystallographic findings suggest a structural basis for these two conformations (Mottonen et al. (1992), supra), their biological significance remains unknown. Negatively-charged phospholipids can convert latent PAI-1 to the active form, suggesting that cell surfaces may modulate PAI-1 activity Lambers et al. (1987) J.Bio.Chem. 262:17492-17496. The observation that latent PAI-1 infused into rabbits is apparently converted to the active form is consistent with this hypothesis Vaughan et al.(1990) Cir.Res. 67:1281-1286. Kinetic and other evidence has also been presented for a second site of interaction between PAI-1 and tPA, outside of the PAI-1 reactive center. Lawrence et al. (1990), supra; Hekman, et al. (1988). Arch.Biochem.Biophys. 262, 199-210.
PAI-1 plays an important role in the fibrinolytic system, in which it reduces the endogenous ability to remove fibrin by inhibiting plasminogen activators (PAs), such as tissue type PA (tPA) and urokinase-type plasminogen activator (uPA). Studies also have shown that elevations of PAI-1 are associated with increased risk for thromboembolic disease. Hamsten et al.(1985) N. Engl. J. Med. 313:1557-1563; Krishnamurti et al. (1992) Semin. Thromb. Hemost. 18:67-80; Schneiderman et al. (1992) Proc. Natl. Acad. Sci. USA 89:6998-7002. Therefore, inactivation of PAI-1 would potentially be of great therapeutic value. Levi et al. (1994) Blood 83: 351-356; Fay et al. (1995) Circulation 91: 1175-1181.
Strategies for reducing PAI activity in vivo fall into two basic categories. First, PAI-1 synthesis can be reduced directly through the action of drugs that depress PAI-1 gene expression. Second, PAI-1 activity can be blocked by specific antibodies or by pharmacological agents that act as specific inhibitors of PAI-1. Direct inactivators of PAI-1 have the potential for specifically reducing the total PAI-1 activity in plasma, and several recent reports demonstrated their efficacy. Levi et al., supra; Biemond et al. (1995) Circulation 91: 1175-1181; Stringer et al. (1994) Arierioscler. Thromb. 14: 1452-1458; Charlton et al. (1997) Fibrinolysis & Proteolysis 11: 51-56; Friderich et al. (1997) Circulation 96: 916-921; van Giezen et al. (1997) Thromb. Haemost 77: 964-969.
In the first studies to use this approach, PAI-1 activity was reduced by reaction with specific anti-PAI-1 monoclonal antibodies. Levi et al., supra; Biemond et al., supra; Stringer et al., supra; Debrock et al. (1997) Biochim. Biophys. Acta (1997) 1337:257-266; Debrock et al. (1997) Gene (1997) 189: 83-88. These studies demonstrate that anti-PAI-1 antibodies can be effective at neutralizing PAI-1 activity both in vitro and in vivo, and suggest that in circumstances when acutely high PAI-1 levels may be detrimental, such therapies may be useful, as for example, as an adjunct to thrombolytic therapy. Although antibody therapy looks promising, its long-term use is unlikely to be successful.
A related strategy for therapy that shows more promise is to prevent or at least influence reactive center loop insertion during PAI-1 interaction with PAs, and thereby prevent formation of the stable covalent complex. This method of blocking PAI-1 activity has been reported to occur in at least two different systems. In the first system, a synthetic peptide analogous to the reactive center loop of PAI-1 inserts into the β-sheet, and once there, it prevents the efficient insertion of the natural loop upon cleavage by a PA. Kvassman et al. (1995) J. Biol. Chem. 270: 27942-27947. As a consequence, PAI-1 is converted to a substrate for PAs, effecting an irreversible inactivation of PAI-1 by a PA. In a second study, a longer peptide of related sequence was also shown to inactivate PAI-1 Eitzman et al. (1995) J. Clin. Invest. 95:2416-2420. But this peptide seemed to induce a conformational change in PAI-1 that did not convert it to a substrate but instead to the non-inhibitory latent form.
Conventional technology has not afforded large scale and relatively simple methodology for selecting potentially effective compounds to treat cardiovascular disease and other pathological conditions that are characterized by elevated levels of a proteinase inhibitor, particularly PAI-1.